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A role for cytosolic fumarate hydratase in urea cycle metabolism and renal neoplasia.

Adam J, Yang M, Bauerschmidt C, Kitagawa M, O'Flaherty L, Maheswaran P, Özkan G, Sahgal N, Baban D, Kato K, Saito K, Iino K, Igarashi K, Stratford M, Pugh C, Tennant DA, Ludwig C, Davies B, Ratcliffe PJ, El-Bahrawy M, Ashrafian H, Soga T, Pollard PJ - Cell Rep (2013)

Bottom Line: On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism.Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls.Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology and Metabolism Group, Nuffield Department of Medicine, Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford OX3 7BN, UK.

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Generation and Analyses of FH-Expressing Transgenic Mice to Investigate the Role of Cytosolic FH/Fumarate in Renal Cyst Development(A) The FH and FHcyt transgenes were cloned into the CB92 vector and targeted to the Rosa26 locus, using phage-mediated recombination (Chen et al., 2011).(B and C) Localization of FH and FHcyt was confirmed in ES cells by immunocytochemistry. Colocalization (yellow) of FH (green) and mitochondria (red) is evident (B), whereas FHcyt is absent from the mitochondria (C). Nuclei (blue) are visualized with DAPI.(D) Kidneys harvested from 30-week-old control, transgene-only (FH, FHcyt), and genetically “rescued” mice (FH1KO+FH and FH1KO+FHcyt) appeared macroscopically normal compared to FH1KO kidneys, which appeared enlarged and cystic.(E) Hematoxylin and eosin (H&E) staining of kidneys harvested from mice at 13, 20, and 30 weeks reveal that transgenic expression of either FH or FHcyt is sufficient to ameliorate cyst development.(F–H) Numbers and frequency of macrocysts (>0.5 mm) and microcysts (>0.1 mm) were determined in each group (n = 6). Error bars represent the SEM.Scale bars, 5 μm (B and C), 10 mm (D), and 100 μm (E). Error bars indicate SEM. See also Figure S1.
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fig2: Generation and Analyses of FH-Expressing Transgenic Mice to Investigate the Role of Cytosolic FH/Fumarate in Renal Cyst Development(A) The FH and FHcyt transgenes were cloned into the CB92 vector and targeted to the Rosa26 locus, using phage-mediated recombination (Chen et al., 2011).(B and C) Localization of FH and FHcyt was confirmed in ES cells by immunocytochemistry. Colocalization (yellow) of FH (green) and mitochondria (red) is evident (B), whereas FHcyt is absent from the mitochondria (C). Nuclei (blue) are visualized with DAPI.(D) Kidneys harvested from 30-week-old control, transgene-only (FH, FHcyt), and genetically “rescued” mice (FH1KO+FH and FH1KO+FHcyt) appeared macroscopically normal compared to FH1KO kidneys, which appeared enlarged and cystic.(E) Hematoxylin and eosin (H&E) staining of kidneys harvested from mice at 13, 20, and 30 weeks reveal that transgenic expression of either FH or FHcyt is sufficient to ameliorate cyst development.(F–H) Numbers and frequency of macrocysts (>0.5 mm) and microcysts (>0.1 mm) were determined in each group (n = 6). Error bars represent the SEM.Scale bars, 5 μm (B and C), 10 mm (D), and 100 μm (E). Error bars indicate SEM. See also Figure S1.

Mentions: Given that the part of the urea cycle affected by fumarate accumulation functions in the cytosol (Shambaugh, 1977), we hypothesized that cytosolic FH may be important in the pathogenesis of HLRCC. Previously, we demonstrated that expression of cytosolic FH in FH1KO MEFs reduced fumarate levels significantly with concomitant loss of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2/NRF2) and hypoxia-inducible factor (HIF) expression, but did not restore defects in oxidative metabolism (Adam et al., 2011; O’Flaherty et al., 2010). To investigate the in vivo role of cytosolic FH, we constructed two transgenic mouse lines stably expressing either FH or FHcyt (excluded from the mitochondria) with a C-terminal V5 affinity tag and under the control of the CAG promoter (Niwa et al., 1991). Equivalent expression between both lines was ensured by targeting the FH transgenes to the Rosa26 locus (Zambrowicz et al., 1997) using integrase-mediated cassette exchange (Chen et al., 2011) (Figures 2A and S1). Targeting fidelity was assessed using PCR (Figure S1), and FH protein localization was confirmed in embryonic stem (ES) cells by immunofluorescence (Figures 2B and 2C). Transgenic expression of FH-V5 was analyzed by immunoblotting and immunofluorescence (Figure S1). Similar to HLRCC patients with renal cancer, mice with kidney-specific FH1 deletion develop hyperplastic renal cysts (Pollard et al., 2007). We intercrossed FH1KO mice with both transgenic lines (FH1KO+FH and FH1KO+FHcyt). Macroscopic analyses of kidneys from 30-week-old mice (Figure 2D) indicated that expression of either transgene was sufficient to ameliorate the increased renal mass in FH1KO mice, and microscopic analysis at three time points (13, 20, and 30 weeks) confirmed that transgenic expression of cytosolic FH was sufficient to suppress cyst development (Figures 2E–2H).


A role for cytosolic fumarate hydratase in urea cycle metabolism and renal neoplasia.

Adam J, Yang M, Bauerschmidt C, Kitagawa M, O'Flaherty L, Maheswaran P, Özkan G, Sahgal N, Baban D, Kato K, Saito K, Iino K, Igarashi K, Stratford M, Pugh C, Tennant DA, Ludwig C, Davies B, Ratcliffe PJ, El-Bahrawy M, Ashrafian H, Soga T, Pollard PJ - Cell Rep (2013)

Generation and Analyses of FH-Expressing Transgenic Mice to Investigate the Role of Cytosolic FH/Fumarate in Renal Cyst Development(A) The FH and FHcyt transgenes were cloned into the CB92 vector and targeted to the Rosa26 locus, using phage-mediated recombination (Chen et al., 2011).(B and C) Localization of FH and FHcyt was confirmed in ES cells by immunocytochemistry. Colocalization (yellow) of FH (green) and mitochondria (red) is evident (B), whereas FHcyt is absent from the mitochondria (C). Nuclei (blue) are visualized with DAPI.(D) Kidneys harvested from 30-week-old control, transgene-only (FH, FHcyt), and genetically “rescued” mice (FH1KO+FH and FH1KO+FHcyt) appeared macroscopically normal compared to FH1KO kidneys, which appeared enlarged and cystic.(E) Hematoxylin and eosin (H&E) staining of kidneys harvested from mice at 13, 20, and 30 weeks reveal that transgenic expression of either FH or FHcyt is sufficient to ameliorate cyst development.(F–H) Numbers and frequency of macrocysts (>0.5 mm) and microcysts (>0.1 mm) were determined in each group (n = 6). Error bars represent the SEM.Scale bars, 5 μm (B and C), 10 mm (D), and 100 μm (E). Error bars indicate SEM. See also Figure S1.
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fig2: Generation and Analyses of FH-Expressing Transgenic Mice to Investigate the Role of Cytosolic FH/Fumarate in Renal Cyst Development(A) The FH and FHcyt transgenes were cloned into the CB92 vector and targeted to the Rosa26 locus, using phage-mediated recombination (Chen et al., 2011).(B and C) Localization of FH and FHcyt was confirmed in ES cells by immunocytochemistry. Colocalization (yellow) of FH (green) and mitochondria (red) is evident (B), whereas FHcyt is absent from the mitochondria (C). Nuclei (blue) are visualized with DAPI.(D) Kidneys harvested from 30-week-old control, transgene-only (FH, FHcyt), and genetically “rescued” mice (FH1KO+FH and FH1KO+FHcyt) appeared macroscopically normal compared to FH1KO kidneys, which appeared enlarged and cystic.(E) Hematoxylin and eosin (H&E) staining of kidneys harvested from mice at 13, 20, and 30 weeks reveal that transgenic expression of either FH or FHcyt is sufficient to ameliorate cyst development.(F–H) Numbers and frequency of macrocysts (>0.5 mm) and microcysts (>0.1 mm) were determined in each group (n = 6). Error bars represent the SEM.Scale bars, 5 μm (B and C), 10 mm (D), and 100 μm (E). Error bars indicate SEM. See also Figure S1.
Mentions: Given that the part of the urea cycle affected by fumarate accumulation functions in the cytosol (Shambaugh, 1977), we hypothesized that cytosolic FH may be important in the pathogenesis of HLRCC. Previously, we demonstrated that expression of cytosolic FH in FH1KO MEFs reduced fumarate levels significantly with concomitant loss of nuclear factor (erythroid-derived 2)-like 2 (NFE2L2/NRF2) and hypoxia-inducible factor (HIF) expression, but did not restore defects in oxidative metabolism (Adam et al., 2011; O’Flaherty et al., 2010). To investigate the in vivo role of cytosolic FH, we constructed two transgenic mouse lines stably expressing either FH or FHcyt (excluded from the mitochondria) with a C-terminal V5 affinity tag and under the control of the CAG promoter (Niwa et al., 1991). Equivalent expression between both lines was ensured by targeting the FH transgenes to the Rosa26 locus (Zambrowicz et al., 1997) using integrase-mediated cassette exchange (Chen et al., 2011) (Figures 2A and S1). Targeting fidelity was assessed using PCR (Figure S1), and FH protein localization was confirmed in embryonic stem (ES) cells by immunofluorescence (Figures 2B and 2C). Transgenic expression of FH-V5 was analyzed by immunoblotting and immunofluorescence (Figure S1). Similar to HLRCC patients with renal cancer, mice with kidney-specific FH1 deletion develop hyperplastic renal cysts (Pollard et al., 2007). We intercrossed FH1KO mice with both transgenic lines (FH1KO+FH and FH1KO+FHcyt). Macroscopic analyses of kidneys from 30-week-old mice (Figure 2D) indicated that expression of either transgene was sufficient to ameliorate the increased renal mass in FH1KO mice, and microscopic analysis at three time points (13, 20, and 30 weeks) confirmed that transgenic expression of cytosolic FH was sufficient to suppress cyst development (Figures 2E–2H).

Bottom Line: On the basis of comprehensive metabolomic analyses, we demonstrate that FH1-deficient cells and tissues exhibit defects in the urea cycle/arginine metabolism.Furthermore, acute arginine depletion significantly reduced the viability of FH1-deficient cells in comparison to controls.Our findings highlight the importance of extramitochondrial metabolic pathways in FH-associated oncogenesis and the urea cycle/arginine metabolism as a potential therapeutic target.

View Article: PubMed Central - PubMed

Affiliation: Cancer Biology and Metabolism Group, Nuffield Department of Medicine, Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford OX3 7BN, UK.

Show MeSH
Related in: MedlinePlus